Through the development of recombinant DNA techniques, it has become fairly straightforward to clone DNA sequences from essentially any organism into plasmid or viral vectors for propagation and amplification in a foreign host. In this form the DNA can be studied with regard to its sequence, structure, coding capacity, or other properties. It can also be used for a variety of applications such as detection of complementary sequences in samples, generation of altered forms of a gene product, modulation of organismal function through insertion into new organisms, etc. Some vectors contain DNA sequences near the insertion site for foreign DNA which control the expression of the inserted DNA in the host cell. These vectors can be used to produce the product of a cloned gene in a host such as Escherichia coli.
More recently it has become possible to efficiently control the expression of cloned DNA sequences in vitro. The first system to be widely exploited for this purpose used a plasmid containing a late promoter from the Salmonella typhymurium bacteriophage SP6 and a purified DNA dependent RNA polymerase purified from the virus infected cells. Krieg, P. A. and Melton, D. A. (1984) Nucleic Acids Res. 12:7057-7070; Butler, E. T. and Chamberlin, M. J. (1982) J. Biol. Chem. 257:5772-5778. Using this system, an RNA copy of one strand of any DNA sequence inserted into the vector can be produced. The vector is constructed such that several unique restriction enzyme sites lie adjacent to the SP6 promoter to allow the insertion of a variety of DNA sequences into that region. The plasmid is then propagated in and purified from E. coli. Next, the purified plasmid is converted to a linear piece of DNA through the action of a restriction enzyme that cuts next to the inserted DNA on the side distal to the promoter. The purified RNA polymerase is added to the linearized DNA along with a substrate mixture and large amounts of the desired RNA can be produced. This RNA can be used as a hybridization probe, as a substrate for RNA processing enzymes, or as mRNA for synthesizing protein by in vitro translation. The relatively large amounts of RNA so produced are readily studied from a variety of structural and functional perspectives.
An analogous system has been configured using a different promoter and RNA polymerase from the E. coli bacteriophage T7. The T7 enzyme recognizes a specific DNA promoter sequence and has similar properties to the SP6 derived enzyme. Both the SP6 and T7 enzymes are extremely specific as they only recognize their own late phage promoters for in vitro transcription. The transcription reactions for either promoter system are very efficient and many copies of full length RNA may be produced from each template molecule. It is thus possible to synthesize milligram amounts of RNA from any cloned DNA sequence.
The above described in vitro transcription systems have the disadvantage that only one strand of the DNA molecule can be copied into RNA. For many applications an RNA copy of a specific strand of the DNA is needed, as for in vitro translation or RNA processing or synthesizing probes for RNA detection. At the time the DNA sequence is cloned into the vector, it often is not known which strand will be required and it often is not possible to control the orientation of the insertion. In such cases, the DNA must be inserted into two different vectors or a number of isolates must be generated and examined to obtain two plasmids which carry the same DNA sequence in opposite orientations with respect to the promoter. For other applications, such as processing double-stranded RNA, both strands of the DNA must be copied into RNA, again requiring much more effort since plasmids with the DNA in two orientations must be isolated.